Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system consisting of three proteins that form a complex in the membrane and a maltose binding protein (MBP) in the periplasm. The interaction of the MBP with the membrane complex stimulates ATP hydrolysis by the complex and allows maltose transport to occur. The maltose proteins belong to a superfamily of transport proteins several of which are medically important, including the proteins responsible for cystic fibrosis and the multi-drug resistance phenotype of tumor cells. The conservation of several structural features in this family, including the presence of two ATP-binding domains; suggests that information learned about the structure and function of one system will be directly applicable to the other systems. The maltose transport system has been purified and reconstituted into phospholipid vesicles in a functional form making it an excellent model system for study. The objective of the proposed research is to understand how the structural and mechanistic features of the maltose system result in active transport. Measurements of ATP-binding and ATP hydrolysis in conjunction with site directed mutagenesis experiments to inactivate one ATP-binding site will be used to determine the role of two ATP-binding sites in hydrolysis. Spectrophotometric approaches will be used to detect different conformational states of the transport complex that are involved in translocation. The reversibility of maltose transport will be studied to determine how ATP hydrolysis supports the accumulation of maltose against a concentration gradient. Finally, isolation of sugar specificity mutants will be used to identify residues in the transport complex which are involved in maltose recognition.